SUMMARY The ultimate expression of the genetic makeup of a cell resides in its proteins. When and how much of a protein will be synthesized depends on RNA molecules, which not only direct the process, but also constitute the machinery which assembles the proteins from amino acids. Thus, understanding RNA metabolism is a prerequisite for elucidating gene expression in normal cells and for clarifying what has gone wrong under a variety of pathological conditions, many of which are manifested by abnormal gene expression. While considerable information has accumulated about the ribonucleases (RNases) essential to RNA metabolism, much remains to be learned about their number, their physiological roles, their mechanism of action, and whether and how they might be regulated. It is the long term goal of this project to define the enzymes, pathways and regulatory processes that constitute the totality of RNA metabolism in a single cell. As RNA metabolism is remarkably similar in all cells, our studies continue to be carried out using the model organism, Escherichia coli, for which a large amount of information is already available. Our specific aims are: 1a) Examination of growth phase regulation of RNase BN and RNase D; 1b) Use the information from Aim 1a to determine how changes in the levels of RNases BN and D regulate small RNAs; 2) Elucidation of the pathways and RNases involved in ribosomal RNA degradation; 3) Determination of the RNases and factors responsible for maturation of ribosomal RNA; and 4) Analysis of the intrinsic helicase activity of RNase R and its importance to the mechanism of action and in vivo functions of RNase R. To accomplish these aims we will use a combination of approaches involving biochemical, molecular biological, genetic, physiological and immunological procedures. We anticipate that the studies detailed in this proposal will provide important, new information on the enzymes, factors and processes that encompass RNA metabolism.